Cell lineage conversion in the sea urchin embryo

The mesoderm of the sea urchin embryo conventionally is divided into two populations of cells; the primary mesenchyme cells (PMCs), which produce the larval skeleton, and the secondary mesenchyme cells (SMCs), which differentiate into a variety of cell types but do not participate in skeletogenesis. In this study we examine the morphogenesis of embryos from which the PMCs have been removed microsurgically. We confirm the observation of Fukushi (1962) that embryos lacking PMCs form a complete skeleton, although in a delayed fashion. We demonstrate by microsurgical and cell marking experiments that the appearance of skeletogenic cells in such PMC-deficient embryos is due exclusively to the conversion of other cells to the PMC phenotype. Time-lapse video recordings of PMC-deficient embryos indicate that the converting cells are a subpopulation of late-ingressing SMCs. The conversion of these cells to the skeletogenic phenotype is accompanied by their de novo expression of cell surface determinants normally unique to PMCs, as shown by binding of wheat germ agglutinin and a PMC-specific monoclonal antibody. Cell transplantation and cell marking experiments have been carried out to determine the number of SMCs that convert when intermediate numbers of PMCs are present in the embryo. These experiments indicate that the number of converting SMCs is inversely proportional to the number of PMCs in the blastocoel. In addition, they show that PMCs and converted SMCs cooperate to produce a skeleton that is correct in both size and configuration. This regulatory system should shed light on the nature of cell-cell interactions that control cell differentiation and on the way in which evolutionary processes modify developmental programs.     

Behavior and Ultrastructure of Primary Mesenchyme Cells at Sessile Site during Termination of Cell Migration in Early Gastrulae

The behavior and ultrastructure of primary mesenchyme cells at two ventrolateral sessile sites in early gastrulae were examined by time-lapse videomicroscopy, scanning electron microscopy, and immunotrans-mission electron microscopy using the sea urchin, Hemicentrotus pulcherrimus and the sand dollar. Clypeaster japonicus . At sessile sites in early gastrulae, PMCs terminated their migration after "touch-and-go" behavior, and even after the termination they retained a pulsatile movement. These behaviors indicate that the termination of PMC migration is not due to deprivation of cell motility nor the establishment of firm adhesion between PMCs and the site. PMCs used short cell processes during migration, and extended longer ones during the early period of migration termination. During the final period of migration at the sessile sites, PMCs extended characteristically thin and long cell processes to the basal lamina. These cell processes, as far as present results indicate, never attach to the blastocoel wall cells through the basal lamina. Thus it is indicated that the primary interaction site for PMCs to terminate their migration is the basal lamina.     

Development of the Basal Lamina and Its Role in Migration and Pattern Formation of Primary Mesenchyme Cells in Sea Urchin Embryos

The development and substructure of the basal lamina and its role in migration and pattern formation of primary mesenchyme cells (PMCs) in normal as well as Li⁺- and Zn⁺⁺-treated embryos of sea urchins were investigated by electron microscopy. Major findings were as follows. 1) Network fibrils appear along the basal surface of the blastular wall by the hatching blastula stage. The area covered with fibrils is restricted to the vegetal hemisphere at earlier stages, but extends to the animal hemisphere as development proceeds. 2) Nonfibrous fuzzy material embeds the fibrils to form a basal lamina, but in places the fibrils project from the basal lamina into the blastocoel. The major components of the fuzzy material were digested by glycosidase, which failed to digest the fibrous components. 3) The fibrils can be classified into two types, one Ca⁺⁺-independent and the other Ca⁺⁺-dependent. PMCs apparently utilize the Ca⁺⁺-indepndent fibrils as a substratum for locomotion. 4) After migration, PMCs accumulate in a specific region to form the PMC pattern. This is formed in the area of greatest concentration of Ca⁺⁺-independent fibrils. 5) PMCs in embryos treated with LiCl, in contrast to normal embryos, accumulate in the animal pole region where the Ca⁺⁺-independent fibrils are markedly concentrated.     

Expression of univin, a TGF-beta growth factor, requires ectoderm-ECM interaction and promotes skeletal growth in the sea urchin embryo

Pl-nectin is an ECM protein located on the apical surface of ectoderm cells of Paracentrotus lividus sea urchin embryo. Inhibition of ECM-ectoderm cell interaction by the addition of McAb to Pl-nectin to the culture causes a dramatic impairment of skeletogenesis, offering a good model for the study of factor(s) involved in skeleton elongation and patterning. We showed that skeleton deficiency was not due to a reduction in the number of PMCs ingressing the blastocoel, but it was correlated with a reduction in the number of Pl-SM30-expressing PMCs. Here, we provide evidence on the involvement of growth factor(s) in skeleton morphogenesis. Skeleton-defective embryos showed a strong reduction in the levels of expression of Pl-univin, a growth factor of the TGF-beta superfamily, which was correlated with an equivalent strong reduction in the levels of Pl-SM30. In contrast, expression levels of Pl-BMP5-7 remained low and constant in both skeleton-defective and normal embryos. Microinjection of horse serum in the blastocoelic cavity of embryos cultured in the presence of the antibody rescued skeleton development. Finally, we found that misexpression of univin is also sufficient to rescue defects in skeleton elongation and SM30 expression caused by McAb to Pl-nectin, suggesting a key role for univin or closely related factor in sea urchin skeleton morphogenesis.     

Localized VEGF signaling from ectoderm to mesenchyme cells controls morphogenesis of the sea urchin embryo skeleton

During development, cell migration plays an important role in morphogenetic processes. The construction of the skeleton of the sea urchin embryo by a small number of cells, the primary mesenchyme cells (PMCs), offers a remarkable model to study cell migration and its involvement in morphogenesis. During gastrulation, PMCs migrate and become positioned along the ectodermal wall following a stereotypical pattern that determines skeleton morphology. Previous studies have shown that interactions between ectoderm and PMCs regulate several aspects of skeletal morphogenesis, but little is known at the molecular level. Here we show that VEGF signaling between ectoderm and PMCs is crucial in this process. The VEGF receptor (VEGFR) is expressed exclusively in PMCs, whereas VEGF expression is restricted to two small areas of the ectoderm, in front of the positions where the ventrolateral PMC clusters that initiate skeletogenesis will form. Overexpression of VEGF leads to skeletal abnormalities, whereas inhibition of VEGF/VEGFR signaling results in incorrect positioning of the PMCs, downregulation of PMC-specific genes and loss of skeleton. We present evidence that localized VEGF acts as both a guidance cue and a differentiation signal, providing a crucial link between the positioning and differentiation of the migrating PMCs and leading to morphogenesis of the embryonic skeleton.     

Commitment and response to inductive signals of primary mesenchyme cells of the sea urchin embryo

In the sea urchin embryo, primary mesenchyme cells (PMC) are committed to produce the larval skeleton, although their behavior and skeleton production are influenced by signals from the embryonic environment. Results from our recent studies showed that perturbation of skeleton development, by interfering with ectoderm-extracellular matrix (ECM) interactions, is linked to a reduction in the gene expression of a transforming growth factor (TGF)-beta growth factor, Pl-univin, suggesting a reduction in the blastocoelic amounts of the protein and its putative involvement in signaling events. In the present study, we examined PMC competence to respond to environmental signals in a validated skeleton perturbation model in Paracentrotus lividus. We found that injection of blastocoelic fluid (BcF), obtained from normal embryos, into the blastocoelic cavity of skeleton-defective embryos rescues skeleton development. In addition, PMC from skeleton-defective embryos transplanted into normal or PMC-less blastula embryos are able to position in correct regions of the blastocoel and to engage spicule elongation and patterning. Taken together, these results demonstrate that PMC commitment to direct skeletogenesis is maintained in skeleton perturbed embryos and confirm the role played by inductive signals in regulating skeleton growth and shape.     

A New Technique for Introducing Anti-Fibronectin Antibodies and Fibronectin-Related Synthetic Peptides into the Blastulae of the Sea Urchin, Clypeaster japonicus.

Migration of primary mesenchyme cells (PMCs) of the sea urchin, Clypeaster japonicus , was examined in vivo by introducing anti-fibronectin (FN) IgG, and FN-related synthetic peptides, Gly-Arg-Gly-Asp-Ser-Pro-Cys, Gly-Arg-Gly-Asp-Ser, and Arg-Gly-Asp-Ser (RGDS) into the blastocoel by a new technique. In this technique the embryos are treated with cytochalasin B (CB) and part of the presumptive PMCs (PPMCs) is removed, leaving a hole in the vegetal plate. Then macromolecules are introduced into the blastocoel through this hole. Their introduction was confirmed by introducing them with polystyrene beads as a marker. The hole closes soon after introduction of the materials, and so these materials remain in the blastocoel. After this treatment, blastulae had fewer PMCs, because of partial loss of PPMCs, but their morphogenesis proceeded normally. Introduction of anti-FN IgG or the synthetic peptides inhibited PMC migration in vivo , and this inhibition was associated with failure of the PMCs to form cell processes. These results indicate that sea urchin PMCs use the RGDS amino acid sequence in the FN molecule for migration in vivo .     

Patterns of cells and extracellular material of the sea urchin Lytechinus variegatus (Echinodermata; Echinoidea) embryo,from hatched blastula to late gastrula

Scanning electron microscopy of six stages of Lytechinus variegatus embryos from hatching through gastrulation reveals changes in the shapes of the ectodermal cells and morphological changes in the extracellular material (ECM) in relation to the locations and migratory activities of mesenchyme cells. The classical optical patterns in the blastular wall (Okazaki patterns) are due to differential orientations of the cells, which bend and extend sheet-like lamellipodia over adjoining cells toward the eventual location of the primary mesenchymal ring. The blastocoelic surfaces of the blastomeres become covered with a thin basal lamina (BL) composed of fibers and nonfibrous material. During primary mesenchyme cell (PMC) ingression, a web-like ECM is located in the blastocoel overlying the amassed PMCs. This ECM becomes sparse in migratory mesenchyme blastulae, and is confined to the animal hemisphere. Localized regions of intertwining basal cell processes in the blastular wall are also present during PMC migration. While a distinct BL is present during early and midgastrulation, blastocoelic ECM is absent. Late gastrulae, on the other hand, have an abundance of blastocoelic ECM concentrated near secondary mesenchyme cell protrusive activity. ECM appearing at both the early mesenchyme and late gastrula stages are probably remnants of degraded BL and intercellular matrix preserved by fixation for SEM. Thus, early mesenchyme ECM is formed of BL material whose degradation is necessary for entry of PMCs into the blastocoel. Late gastrula ECM is apparently a degradation product of BL and intercellular material whose destruction is required for fusion of the gut with oral ectoderm in formation of the mouth.     

Study of larval and adult skeletogenic cells in developing sea urchin larvae

The larval skeleton of sea urchin embryos is formed by primary mesenchyme cells (PMCs). Thereafter, the larvae start feeding and additional arms develop. An adult rudiment that contains spines, tube feet, tests, and other parts of the adult body is formed in the eight-armed larva. The cellular mechanism of the later skeletogenesis and the lineage of the adult skeletogenic cells are not known. In this study, the morphogenesis of larval and adult skeletons during larval development of the sea urchin Hemicentrotus pulcherrimus was investigated by immunostaining cells with PMC-specific monoclonal antibodies, which are useful markers of skeletogenic cells. All spicules and the associated cells in the later larvae were stained with the antibodies. We could observe the initiation of skeletal morphogenesis at each developmental stage and visualize the cellular basis of skeleton formation in whole-mount embryos that possessed an intact morphology. There were some similarities between PMCs and the later skeletogenic cells. Both had a rounded shape with some filopodia, and the antigen expression started just before overt spicule formation. In the later-stage embryos, cells with filopodia and faint antigen expression were observed migrating in the blastocoel or aggregating in the presumptive location of new skeletogenesis.     

Isolation,culture, and differentiation of echinoid primary mesenchyme cells

Summary Methods are described for isolation and culture of primary mesenchyme cells from echinoid embryos. Ninety-five percentpure primary mesenchyme cells were isolated from early gastrulae ofStrongylocentrotus purpuratus, exploiting the biological segregation of these cells within the blastocoel. When cultured, more than 90% of the isolated cells reached the differentiated state, spicule formation, in synchrony with in vivo controls. Isolated primary mesenchyme cells were cultured with and without various cellular and acellular components of normal embryos in order to study the potential involvement of these components in the morphogenesis of the primary mesenchyme. Our data indicate that: 1. primary mesenchyme cells lack the ability to form the annular pattern of the primary mesenchymal ring autonomously; 2. they autonomously produce spicules of a characteristic morphology that differs from that of embryonic spicules; 3. morphogenesis of the primary mesenchyme is not affected by association with embryonic basal lamina, blastocoel matrix, or loosely aggregated epithelial cells, or by close confinement of each set of primary mesenchyme cells within the blastocoelar space; and 4. reaggregated, tightly associated epithelial cells can promote normal primary mesenchyme ring formation, and modify the primary mesenchyme-intrinsic spicule pattern to produce more normal spicule forms.     

Ultrastructure and synthesis of the extracellular matrix of Pisaster ochraceus embryos preserved by freeze substitution

When asteroid embryos cryoprotected with propylene glycol are rapidly frozen in liquid propane and freeze substituted with ethanol, preservation of the cells and extracellular matrix (ECM) is excellent. The basal lamina, although thicker and less well defined than in conventionally fixed embryos, demonstrates a region of decreased density just below the cells that corresponds to the lamina lucida and a lamina densa. The former region is often occupied by fibrous material. In addition, as was previously described in conventionally fixed tissues, the basal lamina of the ectoderm is generally thicker and more substantial than that of the endoderm, reinforcing an earlier suggestion that the structure of the basal lamina is different in different regions of the embryo. The ECM of the blastocoel consists of thin “twig-like” elements that form a loose meshwork evenly distributed throughout the blastocoel. Bundles of 20 nm fibers, located within the meshwork, are oriented parallel to the base of the cells of the stomodeum. In the long axis of the embryo, similar fibers are present in the dorsal aspect of the animal between the stomach and the ectoderm and radiate out from the esophagus crossing the region between it and the ectoderm. Immunocytochemical work with three different monoclonal antibodies shows that glycoprotein molecules, synthesized in the Golgi apparatus, are also secreted here and form part of the matrix structure. The results suggest that the blastocoel is filled with a gel-like material reinforced with bundles of 20-nm fibers. The manner in which the observed arrangement could contribute to the development and maintainence of the shape of the embryo is discussed. J Morphol 232:133–153, 1997. © 1997 Wiley-Liss, Inc.     

Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo

In the indirect developing sea urchin embryo, the primary mesenchyme cells (PMCs) acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth. In this study, we characterize the function of the novel gene strim1, which encodes a tripartite motif-containing (TRIM) protein, that adds to the list of genes constituting the epithelial-mesenchymal signaling network. We report that strim1 is expressed in ectoderm regions adjacent to the bilateral clusters of PMCs and that its misexpression leads to severe skeletal abnormalities. Reciprocally, knock down of strim1 function abrogates PMC positioning and blocks skeletogenesis. Blastomere transplantation experiments establish that the defects in PMC patterning, number and skeletal growth depend upon strim1 misexpression in ectoderm cells. Furthermore, clonal expression of strim1 into knocked down embryos locally restores skeletogenesis. We also provide evidence that the Otp and Pax2/5/8 regulators, as well as FGFA, but not VEGF, ligand act downstream to strim1 in ectoderm cells, and that strim1 triggers the expression of the PMC marker sm30, an ectoderm-signaling dependent gene. We conclude that the strim1 function elicits specific gene expression both in ectoderm cells and PMCs to guide the skeletal biomineralization during morphogenesis.     

Inhibition of cell migration in sea urchin embryos by beta-D-xyloside

This investigation examines the effect of exogenous xylosides on primary mesenchyme cell behavior in Strongylocentrotus purpuratus embryos. In confirmation of studies in some other species the addition of 2 mM p-nitrophenyl-beta-D-xylopyranoside blocks the migration but not the initial ingression of primary mesenchyme cells. The blastocoel matrix of treated embryos appears deficient in a 15- to 30-nm-diameter granular component that is observed extensively on the basal lamina and on filopodia of migrating primary mesenchyme cells in untreated embryos. Other blastocoel components appear unaffected by ultrastructural criteria. The incorporation of 35SO4(2-) per embryo into ethanol precipitates of isolated blastocoel matrices was reduced significantly after xyloside treatment but the distribution of 35SO4(2-) after polyacrylamide gel electrophoresis or the glycosaminoglycan composition was unaffected. Chromatography on Sepharose CL-2B demonstrates a reduction in size of sulfated components of the blastocoel. While over 60% of the 35S-labeled material from the blastocoel of normal mesenchyme blastulae is voided from a Sepharose CL-2B column run in a dissociative solvent, only 10% from xyloside treated embryos is voided. Instead, there is a large included peak with Kav of 0.33. This material is acid soluble but cetylpyridinium chloride precipitable. It apparently consists largely of free glycosaminoglycan chains. Based on analysis of chondroitinase ABC digestion products this material consists of 41% chondroitin-6-sulfate and 58% dermatan sulfate. These results are consistent with a role in cell migration for intact chondroitin sulfate/dermatan sulfate proteoglycans in the sea urchin blastocoel matrix.     

An extracellular fibrillar matrix in gastrulating sea urchin embryos

Fine structural studies of fractured developing sea urchin embryos revealed the existence of a voluminous, fibrillar, extracellular matrix composed of fine filaments, twisting fibers and granules lining the blastocoel of midgastrula embryos. Glycine disaggregated embryos also exhibited this material. The fibrillar matrix is closely associated with the basal lamina of the ectodermal cells of the embryo and histochemical studies suggest it is composed mostly of sulfated glycosaminoglycans. The position of the matrix within the blastocoel as well as its organized association with embryonic cell surfaces is consistent with the hypothesis that it plays a major role in guiding the invaginating archenteron during gastrulation.     

A protein of the basal lamina of the sea urchin embryo

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus . The protein has been named PI-200 K or Hp-200 K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.     

Looking into the sea urchin embryo you can see local cell interactions regulate morphogenesis

The transparent sea urchin embryo provides a laboratory for study of morphogenesis. The calcareous endoskeleton is formed by a syncytium of mesenchyme cells in the blastocoel. The locations of mesenchyme in the blastocoel, the size of the skeleton, and even the branching pattern of the skeletal rods, are governed by interactions with the blastula wall. Now Guss and Ettensohn⁽¹⁾ show that the rate of deposition of CaCO₃ in the skeleton is locally controlled in the mesenchymal syncytium, as is the pattern of expression of three genes involved in skeleton formation. They propose that short range signals emanating from the blastula wall regulate many aspects of the biomineralization process.     

Three cell recognition changes accompany the ingression of sea urchin primary mesenchyme cells

At gastrulation the primary mesenchyme cells of sea urchin embryos lose contact with the extracellular hyaline layer and with neighboring blastomeres as they pass through the basal lamina and enter the blastocoel. This delamination process was examined using a cell-binding assay to follow changes in affinities between mesenchyme cells and their three substrates: hyalin, early gastrula cells, and basal lamina. Sixteen-cell-stage micromeres (the precursors of primary mesenchyme cells), and mesenchyme cells obtained from mesenchyme-blastula-stage embryos were used in conjunction with micromeres raised in culture to intermediate ages. The micromeres exhibited an affinity for hyalin, but the affinity was lost at the time of mesenchyme ingression in vivo. Similarly, micromeres had an affinity for monolayers of gastrula cells but the older mesenchyme cells lost much of their cell-to-cell affinity. Presumptive ectoderm and endoderm cells tested against the gastrula monolayers showed no decrease in binding over the same time interval. When micromeres and primary mesenchyme cells were tested against basal lamina preparations, there was an increase in affinity that was associated with developmental time. Presumptive ectoderm and endoderm cells showed no change in affinity over the same interval. Binding measurements using isolated basal laminar components identified fibronectin as one molecule for which the wandering primary mesenchyme cells acquired a specific affinity. The data indicate that as the presumptive mesenchyme cells leave the vegetal plate of the embryo they lose affinities for hyalin and for neighboring cells, and gain an affinity for fibronectin associated with the basal lamina and extracellular matrix that lines the blastocoel.     

Extracellular matrix of sea urchin and other marine invertebrate embryos

The extracellular matrix surrounding the sea urchin embryo (outer ECM) contains fibers and granules of various sizes which are organized in recognizable patterns as shown by ultrastructural studies, particularly stereoimaging techniques. The use of the ruthenium red method for retaining and staining the ECM, with modifications of the Luft (Anatomical Record 171:347-368, 1971) method for invertebrate embryos, allows for the clarification of certain structures, particularly fiber compaction in the interzonal region, and microvillus-associated bodies. The inner ECM in the sea urchin embryo includes the basal lamina and blastocoel matrix. Stereoimages show that the fibers which are loosely distributed in the blastocoel matrix become compacted around the periphery of the blastocoel to form the basal lamina. The ruthenium red method was also used on a number of marine invertebrate embryos and larvae, representing different phyla, to facilitate comparisons between their surface coats. The similarities observed in the specimens shown suggest that ECMs are widely found on marine invertebrate eggs, embryos, and larvae, and that they resemble vertebrate ECMs and may, therefore, have similar functions.